Allosteric cooperativity between peptide and ATP binding sites on cAMP-dependent protein kinase catalytic subunit was studied kinetically for the reaction of phosphorylation of seven peptide substrates. The allosteric effect was quantified in terms of the interaction factor α by comparing binding effectiveness of a substrate molecule with the free enzyme and with the enzyme complex with another substrate. It was discovered that the magnitude of the allosteric feedback between these binding

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... was governed by the effectiveness of substrate binding, which was varied by using different peptides, and the principle 'better binding: stronger allostery' was formulated. This interrelationship was further formalized in terms of a linear-free-energy relationship b p C p , S K α < at sub-millimolar b K values, changed into negative cooperativity with 1 α > at millimolar b K values. This means that inversion of the cooperative effect was induced by substrate structure, and allostery was used by this enzyme as an additional mechanism to discriminate between substrates, facilitating phosphorylation of good substrates and providing additional protection against phosphorylation of bad substrates. Some implications of this allosteric mechanism on substrate specificity of protein kinases were discussed. Abbreviations: Ala-kemptide -peptide inhibitor LRRAALG; AMPPNP -, -imidoadenosine β γ 5′-triphosphate; kemptidepeptide substrate LRRASLG; protein kinase A -catalytic subunit of cAMP dependent protein kinase. Enzyme: cAMP dependent protein kinase -EC 2.7.11.1.